# Biosynthesis and Reactivity of the Active Site of the FeFe Hydrogenases

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2020 · $91,808

## Abstract

Purchasing a Stopped-flow Spectrometer for Characterization of Reaction Intermediates
 of Hydrogenase Enzyme Models
Stopped-flow spectroscopy has become a major tool in the characterization of reaction
intermediates of synthetic models of the active sites of hydrogenases in this project. This NIH-
supported work focuses on both broad classes of hydrogenases ([FeFe]- and [NiFe]) which are
relevant to the metabolism of pathogens and significant in energy technology relative to hydrogen
production. The goal of the research is to elucidate mechanisms by which these enzymes operate
as well as the pathways by which they are biosynthesized. The PI's group and collaborators have
shown that the binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine
of an [Fe(Cys)(CO)2(CN)] organometallic precursor. This work sets the stage for further
investigation of the H-cluster bioassembly. Very recent work using electron paramagnetic
resonance in conjunction with freeze-quench has identified three intermediates that connect
[Fe(cysteine)(CO)2(CN)] to the H-cluster. We anticipate generating downstream intermediates in
the biosynthesis and evaluating their conversions. We however need tools for monitoring rates.
The proposed stopped-flow spectrometer ideally meets this need.
We have previously been using a stopped-flow spectrometer from Professor Robert B. Gennis
group in the Department of Biochemistry on the same floor of the building with the PI group. This
spectrometer, Applied Photophysics Ltd. SX18.MV, was purchased > 20 years ago and has been
used frequently ever since. The expected instrument lifetime, according to Applied Photophysics
representatives is <15 years, and ours has been heavily used for more than 20 years. Our
instrument has been unreliable for the past few years, and because the instrument is so old and
no longer supported by the company, repairs have been costly, both in parts/labor and in long
delays (often weeks) before the stopped-flow spectrometer can be used for projects. In addition,
the current spectrometer does not have a low temperature control unit. Since most of reaction
intermediates in our hydrogenase models are not stable at room temperature, we need to add
such a unit to the spectrometer. Since kinetic measurement is becoming a main technique that
we use for this NIH project, the unreliability of the stopped-flow spectrometer and costly repairs
have become a huge bottleneck in our projects. Therefore, we are seeking supplemental funding
to replace our outdated and aging instrument with a modern model (AppliedPhotophysics SX20)
and to add a new capability of low temperature control unit in order to increase productivity.

## Key facts

- **NIH application ID:** 10135239
- **Project number:** 3R01GM061153-19S1
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Alison R Fout
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $91,808
- **Award type:** 3
- **Project period:** 2000-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10135239

## Citation

> US National Institutes of Health, RePORTER application 10135239, Biosynthesis and Reactivity of the Active Site of the FeFe Hydrogenases (3R01GM061153-19S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10135239. Licensed CC0.

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